Phosphomimetic RUNX1-4D promotes Mk fate bias in MEPs and the HEL cell line. (A) Schematic of HA-tagged RUNX1-WT, RUNX1-4A, and RUNX1-4D constructs. Four S/T sites downstream of the DNA binding domain are mutated to alanine [4A] or aspartic acid [4D]. HA-tag is at the N-terminus. Additional serine phosphorylation sites are also highlighted. (B) Effects on colony formation of MEPs transduced with transgenes indicated. Each figure represents 3 individual experiments. Colony types as described in Figure 1. Data from individual experiments provided in supplemental Figure 2G. (C) Western blot probed with anti-HA antibody to detect transgenic proteins in transduced HEL cells. (D) Bar graph representing the mean and SD (error bars) of anti-HA western blot data from 3 biological repeats. Band intensities were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) signal in the corresponding lanes, then normalized to RUNX1-WT (dashed line, 1.0). Dots of the same shape represent data from individual experiments. (E) Representative image of CD41/CD42 staining in HEL cells expressing empty vector (EV) or RUNX1-4A/WT/4D without TPA treatment. (F) Graph with CD41⁺CD42⁻ population on the bottom and CD41⁺CD42⁺ on top (n = 5). For panels B,D,F, average ± SD are graphed (∗P < .05, ∗∗∗P < .001, ∗∗∗∗P < .0001). Data from individual experiments provided in supplemental Figure 3D.